Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.     

Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT

The three-dimensional reconstruction of apo-CCT-alpha-actin by cryoelectron microscopy shows that actin binds either the CCTbeta-CCTdelta or the CCTepsilon-CCTdelta subunit pairs of the chaperonin in an open and apparently quasi-native conformation. The CCT-binding sites are seen located at the tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through beta-actin peptide-array screening. Three main CCT binding regions exist: actin Sites I, II, and III, which are composed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on beta-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit reticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, since certain mutants discriminate CCT binding from processing. Actin Site II, located at the tip of actin subdomain 4, is the major determinant for CCT binding. Site II is composed of two anti-parallel extended beta-strands, with F200-T203 and D244 contributing substantially to the binding site. The substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be highly specific to enable capture and folding of the actins and tubulins.     

Platinum carboxylato-pendant-arm macrocycles: structure, redox properties and anti-cancer potential

In an attempt to generate new platinum compounds that may be effective in the treatment of cancer, as well as having a lower toxicity than traditional platins and being orally viable, we are studing the synthesis and reactivity of platinum complexes of tetraazamacrocycles bearing carboxylato pendant arms. We have synthesized adducts of meso- and rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-1,7-diacetic acid (L(1)H(2)). The meso-Pt(II)L(1) complex is unstable with respect to disproportionation, forming platinum metal and [meso-Pt(IV)L(1)](2+). The rac-isomer shows less tendency to disproportionate. Cyclic voltammetry suggests that the rac-Pt(II)L(1) complex undergoes two one-electron oxidations. Using bis-triazacyclononanenickel(III), [Ni(III)(tacn)(2)](3+) as an outer-sphere oxidant, the self-exchange rate for the [Pt(II/III)L](0/+) couple has been estimated at 0.034 M(-1) s(-1).     

Genetic selection for modulators of the MAP kinase and beta-catenin growth-control pathways in mammalian cells

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study

An in vitro model was used to determine the effect of superpulsed CO2 laser energy on normal dermal and keloid-producing fibroblast proliferation and release of growth factors. Growth factors assayed included basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). bFGF is mitogenic, inhibits collagen production, and stabilizes cellular phenotype. TGF-beta1 stimulates growth and collagen secretion and is thought to be integral to keloid formation. Growth in a serum-free medium allowed measurement of these growth factors without confounding variables. Keloid and normal dermal fibroblasts cell lines were established from facial skin samples using standard explant techniques. Samples consisted of three separate keloid and three separate normal dermal fibroblast cell lines. Cells were used at passage 4 to seed 24-well trays at a concentration of 6 x 10(4) cells per milliliter in serum-free medium. At 48 hours, 18.8 percent of each cell well was exposed to a fluence of 2.4, 4.7, and 7.3 J/cm2 using the superpulsed CO2 laser. Cell viability and counts were established at four time points: 0 (time of superpulsed CO2 laser treatment), 24, 72, and 120 hours. Supernatants were collected and assessed for bFGF and TGF-beta1 using a sandwich enzyme immunoassay. All cell lines demonstrated logarithmic growth through 120 hours (conclusion of experiment), with a statistically significant shorter population doubling time for keloid fibroblasts (p < 0.05). Use of the superpulsed CO2 laser shortened population doubling times relative to that of controls; the differences were statistically significant in keloid dermal fibroblasts when fluences of 2.4 and 4.7 J/cm2 were used (p < 0.05 and 0.01, respectively). bFGF was present in greater levels in normal dermal fibroblasts than in keloid dermal fibroblasts. Application of superpulsed CO2 demonstrated a trend toward increased bFGF secretion in both fibroblast types; the increase was significant in the keloid group at 4.7J/cm2. A consistent trend in suppression of TGF-beta1 was seen in both groups exposed to superpulsed CO2, with the maximal effect occurring at 4.7 J/cm2. Serum-free culture sustains logarithmic cell growth and allows growth factor measurement without confounding variables from serum-containing media. Superpulsed CO2 enhances fibroblast replication and seems to stimulate bFGF secretion and to inhibit TGF-beta1 secretion. Given the function of these growth factors, the application of superpulsed CO2 may support normalized wound healing. These findings may explain the beneficial effects of laser resurfacing on a cellular level and support the use of superpulsed CO2 in the management of keloid scar tissue.